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Fingerprinting E. coli communities in Little Lagoon, AL to understand their potential sources

Alice C. Ortmann1,2, Justin D. Liefer1,2, James D. Hatfield3, Hugh L. MacIntyre4

1University of South Alabama, 2Dauphin Island Sea Lab, 3Little Lagoon Preservation Society, 4Dalhousie University.

Abstract

Methods

Little Lagoon Sample Sites

Results: Physical conditions

Results: Salinity and CFU Detection

Results: Amplification of mdh and phoE

Results: E. coli community richness

Conclusions

References

Acknowledgements

Since 2007 the number of fecal coliform bacteria (FCB) in the water column of Little Lagoon, AL has been monitored in a collaboration between the Little Lagoon Preservation Society (LLPS) and researchers at the Dauphin Island Sea Lab (DISL) and Dalhousie University (DU). Over this time period, the concentrations of FCB at five sites within the lagoon have varied, with some samples showing concentrations well above the regulatory threshold of 200 CFU l-1. The source of the FCB remains unknown, with no correlation between the concentration of FCB and any other measured parameter, including temperature, salinity, nutrients or microalgal community composition (see poster by MacIntyre et al). FCB may represent a threat to human health or the heath of other organisms within the lagoon: however until the source of the FCB is determined, it is impossible to develop a long-term management plan (see poster by Hatfield et al).

Because FCB represents a wide range of different types of bacteria, including some that occur naturally within the environment, it is necessary to further identify the organisms present in the water column of Little Lagoon and link their identity to potential sources. One method of identifying bacteria is through the use of DNA fingerprinting. A combination PCR and denaturing gradient gel electrophoresis (DGGE) has been used to generate fingerprints which represent the specific E. coli community present in each sample. Samples were collected during a period when Little Lagoon Pass was closed, when high levels of FCB were detected, and after the pass was re-opened. (The pass was closed in response to oil from the Deepwater Horizon spill.) Two different genes in E. coli have been used for fingerprinting, giving us a clear measure of the diversity of the E. coli present within Little Lagoon. These fingerprints will be compared to fingerprints generated from potential sources of FCB contamination to identify which are the most likely sources of FCB in Little Lagoon.

http://www.micrologylabs.com/:/Pictures/Dish%20B.jpg http://www.envcoglobal.com/files/MO-YSI-85.jpg

Quantify coliforms and E. coli

Measure water parameters

Filter to remove large organisms

Concentrate prokaryote cells

Centrifuge cells to concentrate further

Extract DNA from cells using the UltraClean Soil Kit (MoBio Labs)

Amplify genes with PCR

Fingerprint communities with DGGE

Location of the sample sites in Little Lagoon, AL. Site 5 is located in Gator Lake and not within the lagoon itself. Varying levels of development are found around the lagoon, with little development and a wildlife refuge at the west end. Image from Google Earth.

Gulf of Mexico

Bon Secour Bay

Pass55 44

3322 11

T S DO

S -0.0475

DO -0.3289 0.3668

Day -0.6478 -0.0140 0.2262

Table showing Spearman’s Rho values for physical parameters. Bold indicates significant correlations.

Special thanks to the members of the Little Lagoon Preservation Society, Courtney Metzger, Bart Christiaen, Andrea Hale and Natalie Ortell for their assistance. This work was supported by a grant from the Mississippi-Alabama Sea Grant Consortium to H. MacIntyre, A. Ortmann and K. Park.

Esseili MA, Kassem II and Von Sigler LP (2008) Optimization of DGGE community fingerprinting for characterizing Escherichia coli communities associated with fecal pollution. Water Research 42:4467-4476.Lasalde C Rodriguez R and Toranzos GA (2005). Statistical analyses: Possible reasons for unreliability of source tracking efforts. Applied and Environmental Microbiology 71(8): 4690-4695.Von Sigler LP (2006). Evaluation of denaturing gradient gel electrophoresis to differentiate Escherichia coli populations in secondary environments. Environmental Microbiology 8(10): 1703-1711.

1.Temperature and salinity are both correlated with dissolved oxygen (DO). Temperature decreased over the sample period, but did not differ between sites. Salinity was always lower in Sites 1 and 5. Increases in salinity near the end of the sampling correspond to an opening of the pass to the Gulf of Mexico.

2.The abundance of coliforms and E. coli were correlated with each other, suggesting a common source. Both were negatively correlated with the day of sampling, salinity and DO. Coliforms are facultatively anaerobic and may benefit from lower DO conditions.

3.The E. coli communities based on two different genes are low complexity. For mdh, two different sequences were detected, with one detected in 13 of the 14 samples. For phoE, three difference sequences were detected with one sequences detected in 13 of 14 samples. For each gene there was one sample for which no fingerprint was generated. The high similarity between E. coli communities detected in different samples suggests a common source.

4.There appears to be no correlation between E. coli community composition and the detection of E. coli or coliforms on the plates. Genes from E. coli could be detected even when no colonies were detected on the plates. This is likely due to the larger volume of water analyzed using molecular methods.

9/02 Site39/02 Site28/18 Site39/02 Site19/02 Site59/28 Site19/28 Site28/18 Site59/28 Site38/18 Site18/18 Site49/02 Site49/28 Site48/18 Site2

UPGMA analysis using Dice similarity index shows two outlying samples and three main groups. Group 1 (Red) represents communities with 3 mdh sequences and 1 or 2 phoE sequences. Group 2 (Green) contains samples with 2 mdh sequences and 2 phoE. Group 3 (Blue) communities have only 1 mdh and 1 phoE sequences. There is no apparent relationship between the clusters of E. coli communities and other parameters measured.

Results: Cluster analysis of fingerprints

M 8/18 8/18 8/18 M 9/2 9/2 9/28 9/28 M M 1 3 5 2 5 1 2

Examples of DGGE images. mdh DGGE shows 1 or 2 bands in most samples, phoE has 1, 2 or 3 bands. One sample was negative for each gene (9/2 Site3, mdh and 8/18 Site2, phoE).

M M 9/28 9/28 9/28 9/28 M 9/2 9/2 8/18 M 4 3 2 1 5 1 5

1 2 3 4 5 1 2 3 4 5 1 2 3 4

Aug. 18 Sept. 2 Sept. 28

For each gene, 2 independent PCR reactions containing 10 ng of DNA were carried out. The products were pooled and concentrated. This gel shows 3 μl of pooled product run on an agarose gel. mdh is in the top lanes while phoE is shown in the bottom lanes.

Different intensities of the bands suggest that each sample contained different numbers of E. coli cells.

Coliforms E. coli Other CFUDay -0.3519 -0.3384 -0.1357

Temperature 0.2068 0.0006 0.1925Salinity -0.7545 -0.3709 -0.0286

DO -0.5556 -0.3217 -0.3089Coliforms 0.5048 0.2074

E. coli -0.2197

Table showing Spearman’s Rho values for physical parameters and CFUs detected. The sample day, salinity and DO were correlated with both total coliforms and E. coli. Bold indicates significant correlations.

Plot of CFU/ml of coliforms and E. coli vs salinity. As the salinity decreases below ~10 ppt, the number of both coliforms and E. coli detected using Coliscan Easygel plates (Micrology Laboratories) increases.

For total coliforms, >100 colonies on a plate are represented by 10 000 CFS/100 ml.

Results: CFU Detection